Fig. 4.
TRIM5α interacts with LINE-1 RNPs in the cytoplasm. (A) Human embryonal carcinoma cells 2102EP were probed with ORF1p-specific (red) and TRIM5α-specific antibodies (green) an analyzed by confocal microscopy. Cellular DNA was probed with DAPI. Colocalizing signals are colored in yellow, marked (white arrow) and counted in 25 cells under the microscope. Enlargement: Intensity plots illustrate the colocalization of fluorescent signals in defined areas. (B) HEK 293T cells were transfected with TRIM5α-HA or the nuclear localization signal-containing variant TRIM5α-NLS-HA. Five days posttransfection, GFP-positive cells were quantified by flow cytometry. The frequency of GFP-positive cells is shown as mean of triplicate transfections with errors bars indicating the SD. One out of three independent experiments is shown. Statistical analysis was done using one-way ANOVA followed by Tukey’s post hoc test. ****P < 0.0001, n.s., not significant. (C) HEK 293T cells were transfected with LINE-1 expression plasmid encoding T7 tagged ORF1p (pAD2TE1) and empty vector or the indicated HA-tagged protein. After 24 h, cells were lysed and HA-tagged proteins were precipitated using anti-HA antibodies bound to magnetic beads. cDNA was synthesized from RNPs using MLV-RT and a LEAP primer targeting the polyA of bound mRNAs. Resulting cDNA was amplified by PCR using primers targeting the 3′ end of LINE-1 and the LEAP primer sequence. Protein expression in lysates and precipitates was controlled by immunoblot. (D) HEK 293T cells were transfected with WT LINE-1 (pAD2TE1) or a construct lacking ORF1p expression (L1RPΔneoΔORF1) (44) together with empty vector or TRIM5α-HA. After 24 h, cells were lysed and TRIM5α-HA was precipitated using anti-HA antibodies. LEAP reaction and PCR were performed as in C. Due to the lack of ORF1p in cell lysates, LINE-1 RNA input from cell lysates was analyzed. One out of three independent experiments is shown.